Nodal透過調控缺氧誘發因子促使腦瘤細胞逆分化

神經膠質瘤 (glioma) 是一種具有高度侵襲力及快速生長的腦部腫瘤，其分化程度的好壞與在臨床上的治療及復發有密切的關係。本實驗室先前研究已證實Nodal會增強神經膠質瘤細胞的增生及侵襲能力。另一部分的實驗指出Nodal會調節神經膠質瘤細胞的分化，但詳細機制尚不清楚。因此本篇研究接續探討Nodal是否透過HIF-1α增加神經膠質瘤逆分化 (de-differentiation) 現象，並促使癌幹細胞(cancer stem cell) 的形成。當小鼠原代培養 (primary culture) 的腦部星狀細胞以外緣性的基因重組Nodal (recombinant Nodal, rNodal) 處理後，會造成胚胎幹細胞標記蛋白包括Nanog、Sox2與Oct-4等的蛋白表現。此外，外緣性rNodal能增強神經膠質瘤細胞株 (U87MG) 神經球形成 (neuro sphere) 的能力。當我們轉殖Nodal載體至Nodal表現較低的腦瘤細胞株GBM中，發現Nodal過度表現會造成HIF-1α明顯上升，及細胞逆分化現象。相反的，使用Nodal蛋白的藥理拮抗劑 (antagonist)：SB431542來抑制高表現Nodal的U87MG細胞株的Nodal訊息傳遞路徑，或轉染專一性Nodal小髮夾RNA (shNodal) 至高表現Nodal的U87MG細胞株，結果均會造成HIF-1α表現量下降，且使神經膠質瘤細胞傾向分化良好，亦可以使CD133族群的細胞比例下降。我們透過啟動子報導基因分析法 (Reporter assay) 證明了給予Nodal受體藥理的拮抗劑SB431542，會影響HIF-1α蛋白的轉錄能力。當我們使用專一性HIF-1α小髮夾RNA (shHIF-1α) 到神經膠質瘤細胞株U87MG後，結果神經膠質瘤細胞傾向分化較好。這證明了Nodal會透過使HIF-1α表現量上升而影響神經膠質瘤細胞有分化不良的傾向，並幫助形成神經膠質瘤初始細胞 (glioma initiating cells)。本實驗期望能了解神經膠質瘤幹細胞形成的過程，並藉由從中阻斷形成機制而達到克服在臨床上治療腦瘤的困難，降低腦瘤高抗藥性及增加病患有效治療的機率。

關鍵字

神經膠質瘤；分化；缺氧誘發因子

並列摘要

Glioma is highly invasive and rapidly proliferating maligant brain tumors. The theraputic effect and outcome of gliomas are related to de-differentiation of glioma cells. In a published study, our laboratory has demonstrated that Nodal promoted the tumors growth and invasiveness in human gliomas. The data also implicated that Nodal could regulate the differentiation of glioma cells, but the detail mechanism is unclear. In the present study, we investigated whether Nodal regulates glioma de-differentiation through HIF-1α, and promotes cancer stem cell formation. We found that treatment of mice astrocyte with recombinant Nodal increased the expression of embryonic stem cell proteins, including Nanog、Sox2 and Oct-4. Additionally, recombinat Nodal enhanced neuro sphere formation in U87MG cells. Ectopic expression of Nodal in low Nodal-expressing GBM cells increased HIF-1α expression and resulted in tumor cell de-differenation. Conversely, treatment with the Nodal protein antagonist SB431542, or transfection of Nodal-specific small hairpin RNAs (shNodal) in high Nodal expressing U87MG cells decreased HIF-1α expression, induced cell differentiation, and decreased the population of CD133 + cells. Furthermore, treatment with SB431542 or transfection of HIF-1α specific small hairpin RNA into U87MG cells decreased HIF-1α expression as demonstrated by reporter gene assay. These results suggest that Nodal may promote glioma cancer cells de-differenation and enhance glioma initiating cells proliferation through HIF-1α expression. Understanding the mechanism of glioma cell de-differentiation may provide significant molecular insight into the malignant nature of glioma, which is helpful in developing tactics to exploit glioma therapy.

並列關鍵字

Gliom ； differentiation ； HIF-1α ； Nodal

參考文獻

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Nodal透過調控缺氧誘發因子促使腦瘤細胞逆分化

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